Detection and quantification of male-specific fetal DNA in the serum of pregnant cynomolgus monkeys (Macaca fascicularis)

Abstract

Because of their developmental similarities to humans, nonhuman primates are often used as a model to study fetal development for potential clinical applications in humans. The detection of fetal DNA in maternal plasma or serum offers a source of fetal genetic material for prenatal diagnosis. However, no such data have been reported for cynomolgus monkeys (Macaca fascicularis), an important model in biomedical research. We have developed a specific, highly sensitive PCR system for detecting and quantifying male-specific fetal DNA in pregnant cynomolgus monkeys. We used multiplex quantitative real-time PCR to analyze cell-free DNA in maternal blood serum obtained from 46 pregnant monkeys at gestational weeks 5, 12, and 22. The presence of SRY gene and DYS14 Y chromosomal sequences was determined in 28 monkeys with male-bearing pregnancies. According to confirmation of fetal sex at birth, the probe and primers for detecting the Y chromosomal regions at each time point revealed 100% specificity of the PCR test and no false-positive or false-negative results. Increased levels of the SRY-specific sequences (mean, 4706 copies/mL serum DNA; range, 1731 to 12,625) and DYS14-specific sequences (mean, 54,814 copies/mL serum DNA; range, 4175-131,250 copies) were detected at week 22. The SRY- and DYS14-specific probes appear to be an effective combination of markers in a multiplex PCR system. To our knowledge, this report is the first to describe the detection of cell-free DNA in cynomolgus monkeys.

Figure 1.

Standard curve and detection limits of copy number for quantification of the SRY gene and DYS14 sequences in the TaqMan multiplex PCR assay. Data represent the C_t values of 2 repetitions per test. The linear regression line was obtained by plotting the C_t values against the log₁₀ of the initial quantity of the input plasmid DNA. C_t values for both markers were detected at the following template copies per reaction: 2 × 10⁵ (a), 2 × 10⁴ (b), 2 × 10³ (c), 2 × 10² (d), and 2 × 10¹ (e). No C_t value was detected at 2 × 10⁰ template copies per reaction. (A) SRY gene. (B) DYS14.

Figure 2.

Comparison of the mean copy numbers of Y chromosome sequences (SRY and DYS14) in the maternal circulation of 5 pregnant monkeys continuously monitored during the 3 pregnancy stages. The real-time PCR quantification of copies of (A) SRY and (B) DYS14 sequences (mean from triplicate tests) were plotted against the time course of pregnancy. An increasing number of cell-free DNA copies in individual male-bearing pregnant monkeys (n = 3) were detected at 5, 12, and 22 wk of gestation. (A) SRY gene detection. (B) DYS14 detection. For both markers, the copy numbers in each animal were significantly different between weeks 5 and 12 and between weeks 12 and 22 (*, P < 0.001).